Cancer and viral infections are a serious threat to modern society. Malignant cancerous growths, due to their unique characteristics, pose serious challenges for modern medicine. These characteristics include: uncontrollable cell proliferation resulting in unregulated growth of malignant tissue, an ability to invade local and even remote tissues, lack of differentiation, lack of detectable symptoms and most significantly, the lack of effective therapy and prevention.
Cancer can develop in any tissue of any organ at any age. The etiology of cancer is not clearly defined but mechanisms such as genetic susceptibility, chromosome breakage disorders, viruses, environmental factors and immunologic disorders have all been linked to a malignant cell growth and transformation.
Antineoplastic chemotherapy currently encompasses several groups of drugs including alkylating agents, purine antagonists and antitumor antibiotics. Alkylating agents alkylate cell proteins and nucleic acids preventing cell replication, disrupting cellular metabolism and eventually leading to cell death. Typical alkylating agents are nitrogen mustard, cyclophosphamide and chlorambucil. Toxicities associated with alkylating agents treatment include nausea, vomiting, alopecia, hemorrhagic cystitis, pulmonary fibrosis and an increased risk of development of acute leukemia.
Purine, pyrimidine and folate antagonists are cell cycle and phase specific and, in order to promote an anti-tumor effect, they require cells to be in the cell replication cycle and in the DNA synthesis phase of replication. The purine antagonists such as 6-mercaptopurine or 6-thioguanidine inhibit de novo purine synthesis and interconversion of purines. The pyrimidine antagonists, such as cytarabine, 5-fluorouracil or floxuridine inhibit DNA synthesis by inhibiting deoxycytidylate kinase and DNA polymerase.
Folate antagonists, e.g., methotrexates, bind tightly with the intracellular enzyme dihydrofolate reductase ultimately leading to cell death resulting from an inability to synthesize pyrimidines. Toxicities associated with the use of these compounds include alopecia, myelosuppression, vomiting, nausea, and cerebellar ataxia, among others.
Plant alkaloids such as vincristine, vinblastine or podophyllotoxins etoposide and teniposide generally inhibit mitosis and DNA synthesis and RNA dependent protein synthesis. Toxicities of these drugs are similar to those described above and include myopathy, myelosuppression, peripheral neuropathy, vomiting, nausea and alopecia.
Antitumor antibiotics such as doxorubicin, daunorubicin and actinomycin act as intercalators of DNA, preventing cell replication, inhibiting synthesis of DNA-dependent RNA and inhibiting DNA polymerase. Bleomycin causes scission of DNA and mitomycin acts as inhibitor of DNA synthesis by bifunctional alkylation. Toxicities of these antibiotics are numerous and severe and include necrosis, myelosuppression, anaphylactic reactions, anorexia, dose-dependent cardiotoxicity and pulmonary fibrosis.
Other compounds used for chemotherapeutical treatment of cancer are inorganic ions such as cisplatin, biologic response modifiers such as interferon, enzymes and hormones. All these compounds, similarly to those mentioned above, are accompanied by toxic adverse reactions.
Thus, it would be extremely advantageous to provide safe and non-toxic chemotherapeutic compositions which would effectively inhibit cancer cell proliferation and suppress neoplastic growth. The Merck Manual, 1218-1225 (1987), 15th Ed. Novel synergistic compositions of this invention provide such treatment.
Similar to cancer, the high degree of infectiousness and fast reproduction cycle of viruses within host organisms make viruses a nuisance and a health hazard.
There is no simple treatment of viral diseases. Viruses are not susceptible to antibiotics. The only available treatment of viral diseases is chemotherapy utilizing viral replication inhibitors in host cells. The Merck Manual, 170, 14th Ed. (1982). Examples of these chemical agents are idoxuridine, acyclovir, ribavirin, vidarabine, gancyclovir, adenine arabinoside (ABA-A) and AZT. These, and other viral replication inhibitors, however, are cytotoxic, hepatotoxic, neurotoxic, nephrotoxic and teratogenic. Virus Diseases, 1-6, Crown Publishers, N.Y. (1978).
Human immunodeficiency virus (HIV) infections known as acquired immunodeficiency syndrome (AIDS), presently constitute a worldwide health hazard. HIV infections are almost always fatal due to a weakened immunoresistance, leading to opportunistic infections, malignancies and neurologic lesions.
There is no effective treatment for AIDS other than the treatment of the opportunistic infections, neoplasms and other complications. Available cytostatic (AZT) and antiviral (acyclovir) drugs are extremely toxic and cause severe adverse reactions. In addition, opioid drug addiction coincides with the emergence of AZT resistance, Chuang et al., NIDA Res. Monograph: Problems of Drug Dependence 14:419 (1993), thereby reducing the efficacy of AZT treatment for opioid-addicted AIDS patients.
Thus it would be highly desirable to have available an effective and yet nontoxic treatment of viral diseases, in particular, AIDS, and further for AZT-resistant patients or viral strains.
Cytomegalovirus (CMV), a dangerous co-infection of HIV, is a subgroup of highly infectious viruses having the propensity for remaining latent in man. CMVs are very common among the adult population and as many as 90% of adults have been exposed to and experienced CMV infections. CMVs are normally present in body liquids such as blood, lymph, saliva, urine, feces, milk, etc. CMV infections may cause abortion, stillbirth, postnatal death from hemorrhage, anemia, severe hepatic or CNS damage. Particularly dangerous are CMV infections afflicting AIDS patients, where CMV may cause pulmonary, gastrointestinal or renal complications. There is no specific therapy for CMVs. CMV is resistant to acyclovir, and to other known antiviral drugs.
Thus, it would be extremely advantageous to have available a drug which would effectively inhibit CMV infections.
Recently, a series of highly effective anti-tumor and anti-viral drugs were identified. These drugs include: substituted and unsubstituted 6-amino-1,2-benzopyrones which are the subject of copending U.S. patent application Ser. No. 08/237,969 filed on May 3, 1994, entitled "6-Amino-1,2-Benzopyrones Useful for Treatment of Viral Diseases;" 5-iodo-6-amino-1,2-benzopyrones and 5-iodo-6-nitroso-1,2-benzopyrones which are the subject of copending U.S. patent applications Ser. No. 07/600,593 filed on Oct. 19, 1990 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and Their Metabolites Useful as Cytostatic and Antiviral Agents" and Ser. No. 08/021,989 filed on Feb. 24, 1993 entitled "Novel 5-Iodo-6-Amino-1,2-Benzopyrones and Their Metabolites Useful as Cytostatic Agents;" 3-nitrosobenzamides, 6-nitroso-1,2-benzopyrones and nitroso-1-(2H)-isoquinolinones which are the subject of copending U.S. patent applications Ser. Nos. 07/780,809, 07/893,429 and 07/965,541 filed Oct. 22, 1991, Jun. 4, 1992 and Nov. 2, 1992, respectively, and entitled "Adenosine Diphosphoribose Polymerase Binding Nitroso Aromatic Compounds Useful As Retroviral Inactivating Agents, Anti-retroviral Agents and Anti-tumor Agents;" various iodo-nitro compounds and iodo-nitroso compounds, which are the subject of copending U.S. patent application Ser. No. 08/060,409 filed on May 12, 1993, entitled "Novel Aromatic Nitro Compounds and Their Metabolites Useful as Anti-Viral and Anti-Tumor Agents," and U.S. patent application Ser. No. 08/076,313 filed Jun. 11, 1993, entitled "Novel Aromatic Nitro and Nitroso Compounds and Their Metabolites Useful as Anti-Viral and Anti-Tumor Agents," the disclosures of which are incorporated herein by reference.
These drugs are of remarkably low toxicity, yet highly effective inhibitors of tumors and viral replication in cell cultures. Their therapeutic spectrum appears to be particularly useful for suppression and inhibition of cancer growth and the treatment of viral infections.
The mechanisms of action of poly (ADP ribose) transferase polymerizing ("pADPRT") CCHC-oxidizing ligands such as aromatic C-nitroso compounds have been recently elucidated. C-nitroso ligands exert cellular action on two levels. Kun et al., Biochemie, in press (1994). One level comprises inactivation of pADPRT by zinc ejection. Recently published experiments have shown that aromatic C-nitroso ligands of pADPRT preferentially destabilize one of the two zinc fingers of the enzyme coincidentally with a loss of enzymatic activity but not DNA binding capacity of the protein. Buki et al., FEBS Lett. 290:181-185 (1991). Based on the similarity to results obtained by site-directed mutagenesis, Gradwohl et al., Proc. Natl. Sci. USA 87:2990-2992 (1990), it appears that the primary attack of C-nitroso ligands occurred at zinc finger F1, Buki et al., FEBS Lett. 290:181-185 (1991). 6-nitroso-1,2 benzopyrone ("NOBP") and 3-nitrosobenzamide ("NOBA"), two C-nitroso compounds that inactivate pADPRT at one zinc finger site completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanisms elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium dependent endonuclease. Rice, et al. Proc. Natl. Sci. USA 89:7703-7707 (1992). This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of pADPRT by C-nitroso drugs de-represses the DNA-degrading activity.
In contrast to the "classical" zinc finger structures identified in a large number of transcription factors where the zinc ligands are CCCC or CCHH, Klug and Rhodes, Trends in Biochem. Sci. 12:464-469 (1987), pADPRT contains two zinc chelates that are asymmetrical, i.e. the ligands are CCHC, Gradwohl et al., Proc. Natl. Acad. Sci. 87:2990-2994 (1990). This seemingly minor modification of the chelate has significant chemical consequence with respect to its stability as it exists in the zinc finger within the protein molecule. Buki et al., FEBS Lett. 290:181-185 (1991). Whereas CCCC or CCHH zinc fingers require basic pH and organomercurials to release zinc ion, Giedroc et al., J. Inorg. Biochem. 28:155-169 (1986), zinc ion exchange between the CCHC zinc fingers of pADPRT and external zinc under physiological circumstances, and oxidative destruction of the CCHC chelates by relatively mild oxidizing agents that are made selective by also being ligands of pADPRT has been demonstrated, Buki et al., FEBS Lett. 290:181-185 (1991). This represents a significant biochemical correlation. The F1 finger, Gradwohl et al., Proc. Natl. Acad. Sci. 87:2990-2994 (1990), of pADPRT is first inactivated by the oxidative ligands, resulting in a loss of DNA-stimulated pADPRT activity without major alterations of the DNA binding ability of pADPRT, but excess oxidant destroys both zinc fingers, Buki et al., FEBS Lett. 290:181-185 (1991).
The second level of activity comprises induction of an pADPRT-degrading aminopeptidase that completely digests pADPRT, whereby the pADPRT-binding sites on DNA become available to endonucleolytic degradation. Kun et al., Biochemie, in press (1994). Auto-poly-ADP-ribosylation protects pADPRT from digestion of aminopeptidase, hence inactivation of poly-ADP-ribosylation (as by aromatic C-nitroso ligands) is a prerequisite to proteolytic pADPRT degradation, leading to apoptosis. Therefore, pADPRT CCHC-oxidizing ligand, such as C-nitroso compounds, are effective anti-tumor compounds.
Retroviral nucleocapsid ("NC") proteins and their respective gag precursors from all strains of known retroviruses contain at least one copy of a zinc-binding polypeptide sequence of the type Cys-X.sub.2 -Cys-X.sub.4 -His-X.sub.4 -Cys ("CCHC"), i.e., a zinc finger domain. Henderson et al., Biol. Chem. 256:8400-8406 (1981). This CCHC sequence is essential for maintaining viral infectivity, Gorelick et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988) and Gorelick et al., J. Virol. 64:3207-3211 (1990), therefore, it represents an attractive target for viral chemotherapy. The HIV-1 gag proteins function by specifically binding to the HIV-1 RNA, anchoring it to the cell membrane for budding of viral particles. Meric et al., J. Virol. 63:1558-1658 (1989); Gorelick et al., Proc. Natl. Acad. Sci. USA 85:8420-8424 (1988); Aldovini et al., J. Virol. 64:1920-1926 (1990); and Lever et al., J. Virol. 63:4085-4087 (1989). Site-directed mutagenesis studies demonstrated that modification of Cys or His residues results in defective viral RNA packaging and noninfectious viral particles are formed. Aldovini et al., J. Virol. 64:1920-1926 (1990) and Lever et al., J. Virol. 63:4085-4087 (1989).
Based on the occurrence of CCHC zinc binding sites in both retroviral nucleocapsid and gag-precursor proteins and in poly(ADP-ribose) polymerase it was reasoned that pADPRT CCHC-oxidizing ligands may also have anti-retroviral effects. Recently it was demonstrated that NOBA and NOBP inhibit infection of human immunodeficiency virus HIV-1 in human lymphocytes and also eject zinc from isolated HIV-1 NC zinc fingers and from intact HIV-1 virions. U.S. Ser. No. 0/087,566 filed Jul. 2, 1993; U.S. Ser. No. 08/076,313 filed Jun. 11, 1993; Rice et al., Nature 361:473-475 (1993); Rice et al., Prac. Natl. Acad. Sci. 90:9721-9724 (1993); Chuang et al., FEBS Lett. 326:140-144 (1993); and Wondrak et al., J. Biol. Chem. in press (1994). The zinc-ejected HIV-1 virions exhibit complete loss of infectivity in human lymphocytes. U.S. Ser. No. 0/087,566 filed Jul. 2, 1993 and U.S. Ser. No. 08/076,313 filed Jun. 11, 1993. It was of special interest that the infectious cycle of AZT-resistant rhesus macaque (MMU-23740)-derived SIV was equally inhibited by NOBA, Chuang et al., FEBS Lett. 326:140-144 (1993), an observation that is particularly relevant to opioid drug abusers where addiction coincides with the emergence of AZT resistance, Chuang et al., NIDA Res. Monograph: Problems of Drug Dependence 14:419 (1993). Therefore, the pADPRT CCHC-oxidizing ligands are effective anti-viral compounds.
While these C-nitroso pADPRT CCHC-oxidizing ligands have been found to be quite effective in in vitro tests, they are relatively water insoluble at physiological pH, exhibit limited stability and limited predictability of delivery to the affected cells due to their solubility and stability characteristics. In addition, the relative chemical instability of aromatic C-nitroso pADPRT CCHC-oxidizing ligands towards glutathione ("GSH") is a distinct disadvantage for their direct application in vivo. Recently precursor molecules which generate active C-nitroso compounds in vivo have been prepared to overcome some of these difficulties.
One recently prepared precursor, a cysteine sulfinic adduct of 3-nitrosobenzamide, releases the C-nitroso molecule at weakly basic conditions. Kun et al., U.S. Pat. No. 5,262,564 issued on Nov. 16, 1993. This sulfinic adduct is an effective C-nitroso donor molecule in cell cultures. Id.
As an alternative method, stable precursor C-nitro molecules which serve as pro-drugs for the active C-nitroso pADPRT CCHC-oxidizing ligands were recently prepared. U.S. patent application Ser. No. 08/076,313 filed on Jun. 11, 1993, which is incorporated herein by reference. A prototype of these pro-drugs, 4-iodo-3-nitrobenzamide ("INO.sub.2 BA") has been shown to be stable, having a shelf life of at least one year. Mendeleyev et al., "Chemotherapeutic Activity of 4-Iodo-3-Nitrobenzamide I: Metabolic Reduction to the 3-Nitroso Derivative and Induction of Apoptosis in Tumor Cells in Culture," submitted (1994) (hereinafter "Mendeleyev et al. (1994).")
The aromatic iodo-nitro pro-drugs are slowly but steadily enzymatically reduced within cells to the active aromatic iodo-nitroso compounds such that tumor cell apoptosis or inactivation of viral replication is induced. Id. See also Chuang et al., Biochemical Pharmacology, submitted (1995). These aromatic iodo-nitro compounds thus provide a ready source of in vivo anti-tumor C-nitroso compounds.
On the benzene ring, ortho-iodo-substitution has been shown to facilitate the electrochemical reduction of the nitro group. Fry, The Chemistry of Amino, Nitroso and Nitro Compounds and Their Derivatives. Part I., S. Patai, ed., 319-335, John Wiley and Sons, N.Y. (1982). Recently the fluoro-, chloro- and bromo-analogues of INO.sub.2 BA were tested for anti-tumor activity. These analogues were less effective than INO.sub.2 BA. Mendeleyev et al. (1994). Unsubstituted nitrobenzamides are at least 10-15 times less effective than INO.sub.2 BA. Id. Thus, it is highly probable that the metabolic reduction of aromatic iodo-nitro compounds in cells is activated by the presence of the iodo group ortho to the nitro group. Id.
It is known that glutathione adds to aromatic nitroso compounds to form labile semimercaptal adducts. Eyer, Chem. Biol. Interactions 24:227-239 (1979); Umemoto et al., Chem. Biol. Interactions 68:57-69 (1988); Ellis et al., Chem. Biol. Interactions 82:151-163 (1992). The semimercaptals undergo oxygen atom migration to yield sulfinimides that hydrolyze to the corresponding aromatic amines plus glutathione sulfinic acid. In addition, it has recently been shown that both NOBA and 4-iodo-nitrosobenzamide ("INOBA") are rapidly reduced to the corresponding hydroxyl amines by GSH and ascorbate in in vitro chemical tests. Since many cell types produce GSH, these reduction pathways can quickly reduce the active C-nitroso compounds available in vivo, thereby diminishing the efficacy of the C-nitroso compounds in vivo.
It was thus reasoned that agents which decrease intracellular levels of GSH ("GSH decreasing agents") would decrease the rate at which the active C-nitroso ligands are reduced to the corresponding amines in the cell. Recently, it has been shown that DL-buthionine sulfoximine ("BSO"), an agent that inhibits GSH biosynthesis, Meister, Pharmacology and Therapeutics 51:155-194 (1991), exhibits synergistic apoptosis-inducing potency when administered in combination with pADPRT CCHC-oxidizing ligands in several types of cancer cells. Mendeleyev et al. (1994). BSO also exhibited synergistic anti-viral activity when administered in combination with pADPRT CCHC-oxidizing ligands in cells infected with SIV. Chuang et al., "Chemotherapeutic Activity of 4-Iodo-3-Nitrobenzamide and 5-Iodo-6-Amino-Benzopyrone on Simian Immunodeficiency Virus (SIV) in Cultures of CEM .times.174 Cells," submitted (1994) (hereinafter "Chuang et al. (1994)").
It is therefore a primary object of this invention to provide stable, soluble, non-toxic, highly effective antineoplastic and/or anti-viral synergistic compositions that inactivate pADPRT and NC p7 zinc fingers oxidatively.
pADPRT ligands that oxidize CCHC zinc fingers are not unique inhibitors of pADPRT. Inhibitory ligands of pADPRT that do not attack zinc have been identified. Such a prototype ligand, 6-amino-1,2-benzopyrone has been shown to have anti-tumor effects. Hakam et al., FEBS Lett. 193:1-4 (1991). These ligands bind non-covalently to the nicotinamide site and internal DNA-seeking domains of pADPRT. Bauer et al., manuscript in preparation (1994). In contrast, CCHC-oxidizing ligands also bind non-covalently to the nicotinamide site, but initially attack the F1 zinc finger oxidatively. Id. The respective molecular pharmacological action of both of these types of pADPRT-inhibitory ligands has been studied in a bovine endothelial cell line that has been transfected and transformed with E-ras and, by cellular cloning, the tumorigenic phenotype has been isolated. Id. Synergistic cytocidal effects of pADPRT CCHC-oxidizing ligands in combination with non-covalent pADPRT-inhibitory ligands has been demonstrated. U.S. Ser. No. 08/076,313 filed Jun. 11, 1993.
pADPRT ligands that oxidize CCHC zinc fingers are also not unique as potential anti-HIV drugs at the DNA level. Ligands of pADPRT that do not attack zinc fingers were also shown to be anti-retroviral, Cole et al., Biochem. Biophys. Res. Commun. 180:504-514 (1991), by mechanisms that relate to the drug-induced binding of pADPRT to either the reverse transcriptase template, Buki et al., Biochem. Biophys. Res. Commun. 180:496-503 (1991), or probably to transcription factors required for retroviral synthesis.
A prototypical molecule, 5-iodo-6-amino-1,2-benzopyrone ("IABP") blocked the infectious cycle of HIV as tested in various cell lines, Cole et al., Biochem Biophys. Res. Commun. 180:504-514 (1991), but had no influence on the CCHC zinc fingers of pADPRT or on the p7 NC protein of retroviruses. Thus, reversible inhibition of pADPRT as well as covalent inactivation of pADPRT by zinc ejection can abrogate HIV or SIV infection, in addition to a direct effect of the pADPRT CCHC-oxidizing ligands on the retroviral zinc fingers of the virion p7, Rice et al., Nature 361:473-475 (1993) and Wondrak et al., J. Biol. Chem. 269:21948-21950 (1994), pointing to a hitherto unrecognized role of pADPRT in the life cycle of retroviruses.
Based on this newly recognized role of pADPRT on the retroviral life cycle and the observance of synergy between pADPRT CCHC-oxidizing ligands in combination with non-covalent pADPRT-inhibitory ligands in cancer cells, it was reasoned that non-covalent ligands of pADPRT in combination with CCHC-oxidizing ligands would increase anti-retroviral activity as compared to such ligands acting independently.
It is therefore also a primary objective of this invention to provide non-toxic, highly effective anti-tumor and/or anti-viral synergistic compositions that inactivate pADPRT and NC p7 zinc fingers oxidatively, and that also inhibit pADPRT non-covalently.